US6761884B1 - Vectors including foreign genes and negative selective markers - Google Patents

Vectors including foreign genes and negative selective markers Download PDF

Info

Publication number
US6761884B1
US6761884B1 US08/289,832 US28983294A US6761884B1 US 6761884 B1 US6761884 B1 US 6761884B1 US 28983294 A US28983294 A US 28983294A US 6761884 B1 US6761884 B1 US 6761884B1
Authority
US
United States
Prior art keywords
gene
human cell
vector
cells
therapeutic agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
US08/289,832
Inventor
Michael Blaese
W. French Anderson
Jeanne R. McLachlin
Yawen L. Chiang
Martin Eglitis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
HEALTH AND HUMAN SERVICES NATIONAL INSTITUTES OF HEALTH DEPARTMENT OF UNITED STATES AS REPRESENTED BY SECRETARY
US Department of Health and Human Services
Genetic Therapy Inc
Original Assignee
US Department of Health and Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Health and Human Services filed Critical US Department of Health and Human Services
Priority to US08/289,832 priority Critical patent/US6761884B1/en
Assigned to GENETIC THERAPY, INC. reassignment GENETIC THERAPY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MCLACHLIN, JEANNE R.
Assigned to UNITED STATES OF AMERICA, THE, AS REPRESENTED BY THE DEPARTMENT OF HEALTH AND HUMAN SERVICES reassignment UNITED STATES OF AMERICA, THE, AS REPRESENTED BY THE DEPARTMENT OF HEALTH AND HUMAN SERVICES ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FRENCH, ANDERSON W.
Priority to US08/450,370 priority patent/US5925345A/en
Assigned to HEALTH AND HUMAN SERVICES, NATIONAL INSTITUTES OF HEALTH, DEPARTMENT OF, UNITED STATES, AS REPRESENTED BY THE SECRETARY reassignment HEALTH AND HUMAN SERVICES, NATIONAL INSTITUTES OF HEALTH, DEPARTMENT OF, UNITED STATES, AS REPRESENTED BY THE SECRETARY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BLAESE, R. MICHAEL
Assigned to GENETIC THERAPY, INC. reassignment GENETIC THERAPY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHIANG, YAWEN L.
Assigned to GENETIC THERAPY, INC. reassignment GENETIC THERAPY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: EGLITIS, MARTIN
Application granted granted Critical
Publication of US6761884B1 publication Critical patent/US6761884B1/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to vectors, in particular viral vectors such as retroviral vectors, which include heterologous, or foreign genes. More particularly, this invention relates to vectors including heterologous gene(s) and a negative selective marker.
  • Vectors are useful agents for introducing heterologous, or foreign, gene(s) or DNA into a cell, such as a eukaryotic cell.
  • the heterologous, or foreign gene(s) is controlled by an appropriate promoter.
  • the vector may further include a selectable marker, such as, for example, a neomycin resistance (neo R ) gene, a hygromycin resistance (hygro R ) gene, or a ⁇ -galactosidase ( ⁇ -gal) gene, said marker also being under the control of an appropriate promoter.
  • retroviral vectors examples include prokaryotic vectors, such as bacterial vectors; eukaryotic vectors, including fungal vectors such as yeast vectors; and viral vectors such as DNA virus vectors, RNA virus vectors, and retroviral vectors.
  • Retroviral vectors which have been employed for introducing heterologous, or foreign, genes or DNA into a cell include Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus and Harvey Sarcoma Virus.
  • the term “introducing” as used herein encompasses a variety of methods of introducing heterologous, or foreign, genes or DNA into a cell, such methods including transformation, transduction, transfection, and infection.
  • a vector which includes a heterologous, or foreign gene, and a gene encoding a negative selective marker.
  • the vector which includes the heterologous, or foreign, gene, and the gene encoding a negative selective marker may be a prokaryotic vector, such as a bacterial vector; a eukaryotic vector, such as a fungal vector, examples of which include yeast vectors; or a viral vector such as a DNA viral vector, an RNA viral vector, or a retroviral vector.
  • a prokaryotic vector such as a bacterial vector
  • a eukaryotic vector such as a fungal vector, examples of which include yeast vectors
  • a viral vector such as a DNA viral vector, an RNA viral vector, or a retroviral vector.
  • the vector is a viral vector, and in particular a retroviral vector.
  • retroviral vectors which may be produced to include the heterologous gene and the gene encoding the negative selective marker include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, Avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumour virus.
  • Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells.
  • Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
  • Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.
  • a packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.
  • MoMuLV Moloney murine leukemia virus
  • pPr80 gag another glycosylated protein
  • MoMuSV Moloney murine sarcoma virus
  • the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5′ portion of MoMuSV.
  • the 5′ structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells.
  • Miller has eliminated extra env sequences immediately preceding the 3′ LTR in the LN vector (Miller et al., Biotechniques , 7:980-990, 1989).
  • Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector.
  • Miller, et al. have developed the combination of the pPAM3 plasmid (the packaging-defective helper genome) for expression of retroviral structural proteins together with the LN vector series to make a vector packaging system where the generation of recombinant wild-type retrovirus is reduced to a minimum through the elimination of nearly all sites of recombination between the vector genome and the packaging-defective helper genome (i.e. LN with pPAM3).
  • the retroviral vector may be a Moloney Murine Leukemia Virus of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al. (1987) and Miller, et al. (1989).
  • Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon.
  • the term “mutated” as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.
  • the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the'sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs.
  • Preferred cloning sites are selected from the group consisting of NotI, SnaBI, SalI, and XhoI.
  • the retroviral vector includes each of these cloning sites.
  • a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, SalI, and XhoI located on the retroviral vector.
  • the shuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
  • the shuttle cloning vector may be constructed from a basic “backbone” vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
  • the shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems.
  • the shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria.
  • the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
  • the vectors of the present invention include one or more promoters.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques , Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoters).
  • Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • Heterologous or foreign genes which may be placed into the vectors of the present invention include, but are not limited to genes which encode cytokines or cellular growth factors, such as lymphokines, which are growth factors for lymphocytes.
  • Other examples of foreign genes include, but are not limited to, genes encoding soluble CD4, Factor VIII, Factor IX, ADA, the LDL receptor, ApoE, and ApoC.
  • Suitable promoters which may control the foreign genes include those hereinabove described.
  • the vector also includes a gene encoding a suitable negative selective marker.
  • a negative selective marker is a gene which encodes thymidine kinase, or TK marker. It is to be understood, however, that the scope of the present invention is not to be limited to any specific negative selective marker or markers.
  • the vector of the present invention may be used to transduce packaging cells and to generate infectious viral particles.
  • the infectious viral particles may be used to transduce cells (eg., eukaryotic cells such as mammalian cells).
  • the vector containing the heterologous gene and the gene encoding the negative selective marker may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroportation, the use of liposomes, and CaPO 4 precipitation.
  • the eukaryotic cells may then be administered in vivo to a host as part of a gene therapy procedure.
  • the eukaryotic cells which contain the infectious viral particles, as well as any other cells which are generated that contain the foreign gene and the gene encoding the negative selective marker, may be killed through the in vivo administration of an agent to a host.
  • the selection of a suitable agent is dependent upon the heterologous gene and the gene encoding the negative selective marker contained in the vector.
  • the negative selective marker may be a gene which encodes for thymidine kinase, or TK.
  • a vector which includes a heterologous gene encoding for a lymphokine, and a gene encoding for thymidine kinase as a negative selective marker, is employed to generate infectious viral particles, such infectious viral particles may be used to transfect tumor induced lymphocytes, or TIL cells. Such cells may then be administered to a host. The lymphokine may stimulate the production of tumor cells in the host.
  • ganciclovir may be administered in vivo and preferably by intravenous injection. In the presence of ganciclovir, such transfected tumor induced lymphocytes, and all other cells containing the gene encoding the lymphokine and the gene encoding thymidine kinase, are killed.
  • the vectors of the present invention may be produced from existing vectors through genetic engineering techniques known in the art such that the resulting vector includes a heterologous or foreign gene and a gene encoding a negative selective marker.
  • a vector system for the transduction of cells which comprises a first vector which includes a foreign gene, and a second vector which includes a gene encoding a negative selective marker.
  • first and second vectors may be of the types hereinabove described, and may be constructed through genetic engineering techniques known to those skilled in the art.
  • the foreign gene contained in the first vector may encode for those substances hereinabove described.
  • the gene encoding the negative selective marker may be a gene encoding for thymidine kinase as hereinabove described, or any other negative selective marker.
  • the first and second vectors may be used to tranduce packaging cells through techniques known in the art, and to generate infectious viral particles.
  • infectious viral particles generated from the first and second vectors may then be used to tranduce cells, such as eukaryotic, and in particular, mammalian cells, which then may be administered to a host as part of a gene therapy procedure.
  • FIG. 1 is a schematic of the construction of the pG 1 vector backbone
  • FIG. 2 shows the sequence of the multiple cloning site of the pG 1 plasmid
  • FIG. 3 is a schematic of the pGene plasmid backbone
  • FIG. 4 shows the sequence of the multiple cloning site for the pGene shuttle plasmid
  • FIG. 5 is a schematic of the construction of the plasmid pG1I21SvTK
  • FIG. 6 is a schematic of the construction of the plasmid pG1T12SvTK
  • FIG. 7 is a schematic of the construction of the plasmid pG1T11SvTK.
  • FIG. 8 is a schematic of the construction of the plasmid pG1TKSvN.
  • G1I21SvTk vector which includes a gene for interleukin 2 (IL-2) and a gene for thymidine kinase (TK).
  • IL-2 interleukin 2
  • TK thymidine kinase
  • the GII21SvTk vector is derived from plasmid pG1.
  • the plasmid pG1 was constructed from pLNSX (Palmer et al., Blood , 73:438-445; 1989).
  • the construction strategy for plasmid pG1 is shown in FIG. 1 .
  • the 1.6 kb EcoRI fragment, containing the 5′ Moloney Sarcoma Virus (MoMuSV) LTR, and the 3.0 kb EcoRI/ClaI fragment, containing the 3′ LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated separately.
  • pG1 consists of a retroviral vector backbone composed of a 5′ portion derived from MoMuSV, a short portion of gag in which the authentic ATG start codon has been mutated to TAG (Bender et al.
  • MCS 54 base pair multiple cloning site
  • the MCS was designed to generate a maximum number of unique insertion sites, based on a screen of non-cutting restriction enzymes of the pG1 plasmid, the neo r gene, the ⁇ -galactosidase gene, the hygromycin r gene, and the SV40 promoter.
  • the pGene plasmid (FIG. 3) does not exist as an independent molecular entity, but rather may be considered a construction intermediate in the process of cloning genes for subsequent insertion into pG1.
  • the basic backbone is that of pBR322 (Bolivar et al., Gene , 2:95 (1977)).
  • These linkers synthesized using an oligo-nucleotide synthesizer, contain a total of 14 unique restriction enzyme recognition sites, as well as sequences felt to enhance mRNA stability and translatability in eukaryotic cells.
  • restriction sites were chosen based on a screen of non-cutting restriction enzymes of the plasmid backbone, the neo r gene, the ⁇ -galactosidase gene, the hygromycin r gene, and the SV40 promoter. (The choice of enzymes results in the sites of pG1 being a subset of the sites in pGene). Genes can be ligated into this backbone with NcoI and XhoI ends.
  • the resulting backbone is roughly 2.1 kb in size and contains a 99 base pair multiple cloning site containing, from 5′ to 3′, the following restriction enzyme recognition sites: SphI, NotI, SnaBI, SalI, SacII, AccI, NruI, BgII, NcoI, XhoI, HindIII, ApaI, and SmaI. (FIG. 4 ). From the BgII to the NcoI sites lies a 27 base pair region containing an mRNA signal based on the work of Hagenbuchle et al. Cell , 13:551-563 (1978).
  • telomere sequence was first constructed.
  • TK Herpes thymidine kinase
  • the sequence of the 5′ EcoRI site was mutated, maintaining amino acid coding fidelity but eliminating the internal EcoRI site to permit directional cloning and screening of the total linkered lacZ fragment into the 2.1 kb NdeI/EcoRI of pBR322.
  • the pBg plasmid was then used to construct an SV40 promoted TK gene.
  • the plasmid pG1I21SvTk was prepared as follows.
  • the IL-2 gene was derived from the plasmid HT-5.1 (ATCC #59396).
  • the 1.0 kb BamHI fragment was isolated from this plasmid and then truncated down to a 445 base pair HgiAI/DraI fragment.
  • a 100 base pair linker was constructed including the entire 20 amino acid coding region of the amino-terminal end of IL-2, and then a 40 base pair stretch identical in sequence to that of pGene between the BglII and NcoI sites is added as a 5′ leader.
  • a SnaBI site was added 5′ to the BglII, permitting direct insertion of this reconstructed IL-2 fragment into pBg which has been digested with SnaBI and HindIII (the HindIII blunted with the Klenow polymerase). From this resulting pI21 plasmid, a 550 base pair BglII/ClaI fragment was isolated blunted, and then inserted into Not I digested and blunted pG1XSvTk.
  • Producer cells for vectors carrying the Herpes simplex virus thymidine kinase gene (TK) as a marker may be generated in two ways. Both methods are developed to generate producer cells containing only a single, provirally integrated vector within the chromosomes of the packaging cell. In the first, the TK gene is co-transfected into PE501 packaging cells (Miller and Rosman, Biotechniques 7:980-990 (1989) with an alternate selectable plasmid, such as pSv2Neo.
  • the PE501 cells are mixed with PA317 amphotropic packaging cells made hygromycin resistant (hygro r ) with a hygromycin phosphotransferase expressing plasmid, and allowed to culture together for 10-14 days.
  • PA317 amphotropic packaging cells made hygromycin resistant (hygro r ) with a hygromycin phosphotransferase expressing plasmid, and allowed to culture together for 10-14 days.
  • the PA317 cells, infected with the TK vector packaged in the PE501 cells are recovered by hygromycin selection.
  • Individual hygro r clones are isolated and evaluated for expression of the non-selectable gene (in the above example IL-2), as well as for their ability to transduce target cells.
  • TK- NIH 3T3 fibroblasts are measured either by the level of non-selectable gene activity that is introduced in target cells, or by direct TK titering using HAT selection (Littlefield, Science 145:709-710 (1964)) of TK- NIH 3T3 fibroblasts in a manner similar to conventional titering with G418 for the neo r gene (Eglitis et al, Science 230:1395-1398 (1985)). Briefly, TK- NIH 3T3 cells are plated at a density of 2 ⁇ 10 4 cells per 35 mm dish and the following day infected for 2-4 hours with various dilutions of virus supernatant containing 8 ⁇ g/ml polybrene.
  • the cells are allowed to grow for an additional 24-48 hours following infection and then were grown in selective medium containing HAT for 10-12 days prior to staining with methylene blue and counting individual HAT resistant colonies.
  • Producer clones which generate between 5 ⁇ 10 4 and 5 ⁇ 10 5 HAT resistant colony-forming units per ml are then used for further evaluation of efficacy.
  • a second method utilizes amphotropic packaging cells which are TK-. Having packaging cells which are TK- is important to generate easily trans-infected producer clones of vectors which only contain the TK gene as selectable marker. It is desired to incorporate packaging functions into cells lacking endogenous TK activity so that when TK vector is introduced the resulting vector-containing cells may be directly selected in HAT medium.
  • a means of accomplishing this is to use the pPAM3 packaging system of Miller et al., Mol. Cell. Biol ., 5:431-437 (1985).
  • the pPAM3 plasmid, encoding for gag, pol, and env is transfected into TK- NIH 3T3 cells (Chen and Okayama, Mol. Cell.
  • This example describes the construction of pG1T12SvTK, which contains a gene encoding TNF- ⁇ .
  • a schematic of the construction of pG1T12SvTK is shown in FIG. 6 .
  • Plasmid pG1XSvTK was constructed as hereinabove described in Example 1. Then, the plasmid pLset5SN was constructed. Plasmid pLset5SN contains a 521 bp NcoI/EcoRI fragment from pTNF-A.BEC (Beckman Catalogue number 267430, and as published in Pennica, et al., Nature , Vol. 312, pg. 724 (1984)) to which had been fused a 51 bp EcoRI/NruI oligomer encoding the natural secretion signal of TNF- ⁇ .
  • a set of 10 oligonucleotides was synthesized and assembled in sets of two or four.
  • the sequence of these oligonucleotides was formulated using the human TNF- ⁇ sequence found in Genbank (Accession number X02159).
  • Genbank accesion number X02159.
  • the oligomers were assembled and ligated to the 5′ end of the 521 bp TNF- ⁇ fragment from pTNF-A.BEC using the pLXSN plasmid (Miller, et al., Biotechniques , Vol. 7, pgs. 980-991 (1990)).
  • pLSET5SN A series of plasmid intermediates, pLSET1SN and pLSET3SN AND LSET4SN, each containing greater assemblages of oligonucleotide fragments, were constructed.
  • pLSET5SN To construct pLSET5SN, an 851 bp HindIII fragment from pLSET4SN containing the majority of the TNF- ⁇ coding sequence was cloned into the unique HindIII site of pLSET3SN immediately following the assembled sequence for the secretion signal. This ligation resulted in the formation of plasmid pLSET5SN, which contains the entire authentic cDNA sequence of TNF- ⁇ , including the sequence of the secretion signal.
  • TNF- ⁇ gene containing the natural secretion signal was then removed from pLset5SN as a 730 bp BglII/Bam HI fragment. This fragment was then blunted using the Klenow fragment, and inserted into the unique SnaBI site of pG1XSvTk, to create the final construct pG1T12SvTk.
  • This example describes the construction of pG1T11SvTk, which, like pG1T12SvTk, contains a gene encoding TNF- ⁇ .
  • a schematic of the construction of pG1T11SvTk is shown in FIG. 7 .
  • Plasmid pG1XSvTk was constructed as described in Example 1. Then the plasmid pT11 was constructed. The same 521 bp NcoI/EcoRI fragment from pTNF-A.BEC is inserted in place of the lacZ gene in NcoI/EcoRI digested pBG, and an 87 base pair oligomer containing the rat growth hormone secretion signal, as formulated using the rat growth hormone sequence found in Genbank (Accession number J00739; Barta, et al., Proc. Natl. Acad. Sci. U.S.A ., Vol. 78, pgs. 4867-4871 (1981)) is then inserted into the NcoI site.
  • the resulting gene is removed as a 608 bp BglII/BamHI fragment, blunted with the Klenow fragment, and inserted into the unique SnaBI site of pG1XSvTk to create pG1T11SvTk.
  • This example describes the construction of pG1TkSvN, a schematic of which is shown in FIG. 8 .
  • This vector contains a gene, under the control of an SV40 promoter, which encodes neomycin resistance.
  • Plasmids pG1 and pBg were constructed as described in Example 1. A 1.74 kb BglII/PvuII fragment was excised from the pX1 plasmid, blunted with the large (Klenow) fragment of DNA polymerase I, and inserted into the unique SnaBI site in the pG1 multiple cloning site, to form plasmid pG1TK.
  • a 339 bp PvuII/HindIII SV40 early promoter fragment was then inserted into pBg in the unique NruI site to generate the plasmid pSvBg.
  • the pSvBg plasmid was digested with BglII/XhoI to remove the lacZ gene, and the ends were made blunt using the Klenow fragment.
  • An 852 bp EcoRI/AsuII fragment containing the coding sequence of the neomycin resistance gene was removed from pN2 (Armentano, et al., J. Virol ., Vol. 61, pgs.
  • This example employs a clone (WP4) (Asher et al., J. Inmunol , Vol. 146, pg. 3227 (1991)) of a murine tumor cell line MCA205 (Wexler et al., J. Natl. Canc. Inst ., Vol. 63, pg. 1393 (1979)) for assessing the utility of the HSV-TK gene in a “suicide” system.
  • WP4 clone
  • WP4 cells are plated in 100 mm tissue culture plates at 1-5 ⁇ 10 5 cells per plate. After 24 hrs. of culture, growth medium is removed and 10 ml of retroviral supernatant containing pG1TKSvN (10 4 -10 5 titer) with 8 ug/ml polybrene is added per plate. The cells are incubated from 18 to 24 hrs., and the supernatant is then removed and the cells are placed in growth medium for an additional 48 hours of culture. At the end of this period, the cells are from 80% to 90% confluent. Each 100 mm plate is split into three 100 mm paltes, and the cells are grown as follows:
  • Plate 1 Expand cells and freeze with no further treatment.
  • Plate 2 Cells are maintained without treatment as an “unselected” population.
  • Plate 3 One day after splitting, the cells are placed in 0.4-0.8 mg/ml G418, and the cells are observed daily. When 60% to 70% of the cells have been killed, the selection medium is removed, the culture is washed in PBS, and growth medium is added. The cells are cultured for another 5 to 7 days. Cultures which are 50% to 60% confluent are prepared, and 0.4-0.8 mg/ml of G418 in growth medium is added. The cells are then monitored for growth. After initial cell death, the cells can be maintained in G418 continuously. These cells represent the “selected” population.
  • Transduced “selected” cells are then plated in 24-well tissue culture plate at 1-2.5 ⁇ 10 4 cells per well. After 24 hrs. of culture, ganciclovir is added in concentrations ranging from 0.01-100 uM, the cells are monitored for growth and all wells are stained in methylene blue when the control wells (no ganciclovir added) are confluent (at 5 to 7 days). Complete killing of transduced selected cells is observed at concentrations from 0.1 to 1.0 uM of ganciclovir, while untransduced cells are killed at concentrations of 100 uM or greater.
  • WP4 tumor cells form tumors in C57BL/6 or BNX mice when injected subcutaneously.
  • the number of cells required for tumor formation will vary depending of the strain of mouse used.
  • C57BL/6 mice require 5 ⁇ 10 6 -1 ⁇ 10 7 cells for tumor formation in 10-14 days.
  • BNX mice require 1-5 ⁇ 10 4 cells for tumor formation in 10-14 days.
  • Tumor cells are suspended in PBS and injected subcutaneously into the hind flank in a 0.5 ml volume.
  • Five to ten days after tumor inoculation, ganciclovir is administered intraperitoneally twice daily for 5 days.
  • a 2-5 mg/ml solution of ganciclovir prepared in serum-free medium (RPMI 1640, DMEM, or other) is used to achieve a dose of 125-150 mg/kg. Animals are monitored and tumor measurements made twice weekly.
  • tumors are palpable, but unmeasurable. Tumors arising from untransduced WP4 cells reach a size of 2.0-2.5 cm 3 in 40-45 days with or without ganciclovir administration. Tumor cells containing the HSV-TK gene initially form palpable tumors then regress completely and become undetectable within 10-20 days after the start of ganciclovir treatment. Complete, long term tumor ablation is observed in 60-80% of mice having received selected cells plus ganciclovir therapy.
  • nucleic acid single linear plasmid DNA 1 AATTCGCGGC CGCTACGTAG TCGACGGATC CCTCCAGAAG CTTGGGCCCA T 51 94 base pairs nucleic acid single linear plasmid DNA Gene may be inserted between base 73 and base 74. 2 CGCATGGCGG CCGCTACGTA GTCGACCCGC GGGTCGACTC GCGAAGATCT TTCCGCAGCA 60 GCCGCCACCA TGGCTCGAGA AGCTTGGGCC CGGG 94

Abstract

A vector, in particular a retroviral vector, which includes a heterologous or foreign gene and a gene encoding a negative selective marker. The negative selective marker enables one to kill cells which contain the gene encoding the negative selective marker, when a particular agent is administered to such cells.

Description

This application is a continuation of application Ser. No 07/792,281, filed Nov. 14, 1991, abandoned.
This invention relates to vectors, in particular viral vectors such as retroviral vectors, which include heterologous, or foreign genes. More particularly, this invention relates to vectors including heterologous gene(s) and a negative selective marker.
Vectors are useful agents for introducing heterologous, or foreign, gene(s) or DNA into a cell, such as a eukaryotic cell. The heterologous, or foreign gene(s) is controlled by an appropriate promoter. In addition, the vector may further include a selectable marker, such as, for example, a neomycin resistance (neoR) gene, a hygromycin resistance (hygroR) gene, or a β-galactosidase (β-gal) gene, said marker also being under the control of an appropriate promoter. Examples of such vectors include prokaryotic vectors, such as bacterial vectors; eukaryotic vectors, including fungal vectors such as yeast vectors; and viral vectors such as DNA virus vectors, RNA virus vectors, and retroviral vectors. Retroviral vectors which have been employed for introducing heterologous, or foreign, genes or DNA into a cell include Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus and Harvey Sarcoma Virus. The term “introducing” as used herein encompasses a variety of methods of introducing heterologous, or foreign, genes or DNA into a cell, such methods including transformation, transduction, transfection, and infection.
In accordance with an aspect of the present invention, there is provided a vector which includes a heterologous, or foreign gene, and a gene encoding a negative selective marker.
The vector which includes the heterologous, or foreign, gene, and the gene encoding a negative selective marker, may be a prokaryotic vector, such as a bacterial vector; a eukaryotic vector, such as a fungal vector, examples of which include yeast vectors; or a viral vector such as a DNA viral vector, an RNA viral vector, or a retroviral vector.
In a preferred embodiment, the vector is a viral vector, and in particular a retroviral vector. Examples of retroviral vectors which may be produced to include the heterologous gene and the gene encoding the negative selective marker include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, Avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumour virus.
Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells. Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
These new genes have been incorporated into the proviral backbone in several general ways. The most straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences within the long terminal repeat (LTR). Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.
Efforts have been directed at minimizing the viral component of the viral backbone, largely in an effort to reduce the chance for recombination between the vector and the packaging-defective helper virus within packaging cells. A packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.
Bender et al., J. Virol. 61:1639-1649 (1987) have described a series of vectors, based on the N2 vector (Armentano, et al., J. Virol., 61:1647-1650) containing a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems. These changes have also reduced the likelihood that viral proteins would be expressed. In the first of these vectors, LNL-XHC, there was altered, by site-directed mutagenesis, the natural ATG start codon of gag to TAG, thereby eliminating unintended protein synthesis from that point. In Moloney murine leukemia virus (MoMuLV), 5′ to the authentic gag start, an open reading frame exists which permits expression of another glycosylated protein (pPr80gag). Moloney murine sarcoma virus (MoMuSV) has alterations in this 5′ region, including a frameshift and loss of glycosylation sites, which obviate potential expression of the amino terminus of pPr80gag. Therefore, the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5′ portion of MoMuSV. The 5′ structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells. In a final alteration to reduce overlap with packaging-defective helper virus, Miller has eliminated extra env sequences immediately preceding the 3′ LTR in the LN vector (Miller et al., Biotechniques, 7:980-990, 1989).
The paramount need that must be satisfied by any gene transfer system for its application to gene therapy is safety. Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector. Miller, et al. have developed the combination of the pPAM3 plasmid (the packaging-defective helper genome) for expression of retroviral structural proteins together with the LN vector series to make a vector packaging system where the generation of recombinant wild-type retrovirus is reduced to a minimum through the elimination of nearly all sites of recombination between the vector genome and the packaging-defective helper genome (i.e. LN with pPAM3).
In one embodiment, the retroviral vector may be a Moloney Murine Leukemia Virus of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al. (1987) and Miller, et al. (1989). Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon. The term “mutated” as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.
In another embodiment, the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the'sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs. Preferred cloning sites are selected from the group consisting of NotI, SnaBI, SalI, and XhoI. In a preferred embodiment, the retroviral vector includes each of these cloning sites.
When a retroviral vector including such cloning sites is employed, there may also be provided a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, SalI, and XhoI located on the retroviral vector. The shuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
The shuttle cloning vector may be constructed from a basic “backbone” vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
The shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems. The shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria. Thus, for example, the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
The vectors of the present invention include one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
Heterologous or foreign genes which may be placed into the vectors of the present invention include, but are not limited to genes which encode cytokines or cellular growth factors, such as lymphokines, which are growth factors for lymphocytes. Other examples of foreign genes include, but are not limited to, genes encoding soluble CD4, Factor VIII, Factor IX, ADA, the LDL receptor, ApoE, and ApoC.
Suitable promoters which may control the foreign genes include those hereinabove described.
The vector also includes a gene encoding a suitable negative selective marker. An example of a negative selective marker is a gene which encodes thymidine kinase, or TK marker. It is to be understood, however, that the scope of the present invention is not to be limited to any specific negative selective marker or markers.
The vector of the present invention may be used to transduce packaging cells and to generate infectious viral particles. The infectious viral particles may be used to transduce cells (eg., eukaryotic cells such as mammalian cells). The vector containing the heterologous gene and the gene encoding the negative selective marker may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroportation, the use of liposomes, and CaPO4 precipitation. The eukaryotic cells may then be administered in vivo to a host as part of a gene therapy procedure. The eukaryotic cells which contain the infectious viral particles, as well as any other cells which are generated that contain the foreign gene and the gene encoding the negative selective marker, may be killed through the in vivo administration of an agent to a host. The selection of a suitable agent is dependent upon the heterologous gene and the gene encoding the negative selective marker contained in the vector.
For example, when the heterologous gene encodes a lymphokine, the negative selective marker may be a gene which encodes for thymidine kinase, or TK. When, for example, such a vector, which includes a heterologous gene encoding for a lymphokine, and a gene encoding for thymidine kinase as a negative selective marker, is employed to generate infectious viral particles, such infectious viral particles may be used to transfect tumor induced lymphocytes, or TIL cells. Such cells may then be administered to a host. The lymphokine may stimulate the production of tumor cells in the host. To prevent the further generation of cells containing the gene encoding the lymphokine and thus preventing the production of tumor cells, one may administer ganciclovir to the host. The ganciclovir may be administered in vivo and preferably by intravenous injection. In the presence of ganciclovir, such transfected tumor induced lymphocytes, and all other cells containing the gene encoding the lymphokine and the gene encoding thymidine kinase, are killed.
The vectors of the present invention may be produced from existing vectors through genetic engineering techniques known in the art such that the resulting vector includes a heterologous or foreign gene and a gene encoding a negative selective marker.
In accordance with an alternative aspect of the present invention, there is provided a vector system for the transduction of cells (e.g., eukaryotic cells and in particular mammalian cells) which comprises a first vector which includes a foreign gene, and a second vector which includes a gene encoding a negative selective marker. Each of the first and second vectors may be of the types hereinabove described, and may be constructed through genetic engineering techniques known to those skilled in the art. The foreign gene contained in the first vector may encode for those substances hereinabove described. The gene encoding the negative selective marker may be a gene encoding for thymidine kinase as hereinabove described, or any other negative selective marker. The first and second vectors may be used to tranduce packaging cells through techniques known in the art, and to generate infectious viral particles. The infectious viral particles generated from the first and second vectors may then be used to tranduce cells, such as eukaryotic, and in particular, mammalian cells, which then may be administered to a host as part of a gene therapy procedure.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention now will be described with respect to the drawings, wherein:
FIG. 1 is a schematic of the construction of the pG1 vector backbone;
FIG. 2 shows the sequence of the multiple cloning site of the pG1 plasmid;
FIG. 3 is a schematic of the pGene plasmid backbone;
FIG. 4 shows the sequence of the multiple cloning site for the pGene shuttle plasmid;
FIG. 5 is a schematic of the construction of the plasmid pG1I21SvTK;
FIG. 6 is a schematic of the construction of the plasmid pG1T12SvTK;
FIG. 7 is a schematic of the construction of the plasmid pG1T11SvTK; and
FIG. 8 is a schematic of the construction of the plasmid pG1TKSvN.
The invention will now be described with respect to the following example; however, the scope of the present invention is not intended to be limited thereby.
EXAMPLE
Construction of TK/IL-2 (Interleukin-2) Vector
The following describes the construction of the G1I21SvTk vector, which includes a gene for interleukin 2 (IL-2) and a gene for thymidine kinase (TK).
The GII21SvTk vector is derived from plasmid pG1. The plasmid pG1 was constructed from pLNSX (Palmer et al., Blood, 73:438-445; 1989). The construction strategy for plasmid pG1 is shown in FIG. 1. The 1.6 kb EcoRI fragment, containing the 5′ Moloney Sarcoma Virus (MoMuSV) LTR, and the 3.0 kb EcoRI/ClaI fragment, containing the 3′ LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated separately. A linker containing seven unique cloning sites was then used to close the EcoRI/ClaI fragment on itself, thus generating the plasmid pGO. The plasmid pGO was used to generate the vector plasmid pG1 by the insertion of the 1.6 kb EcoRI fragment containing the 5′ LTR into the unique EcoRI site of pGO. Thus, pG1 consists of a retroviral vector backbone composed of a 5′ portion derived from MoMuSV, a short portion of gag in which the authentic ATG start codon has been mutated to TAG (Bender et al. 1987), a 54 base pair multiple cloning site (MCS) containing, from 5′ to 3′ the sites EcoRI, NotI, SnaBI, SalI, BamHI, XhoI, HindII, ApaI, and ClaI, and a 3′ portion of MoMuLV from base pairs 7764 to 7813 (numbered as described (Van Beveren et al., Cold Spring Harbor, Vol. 2, pg. 567, 1985) (FIG. 2). The MCS was designed to generate a maximum number of unique insertion sites, based on a screen of non-cutting restriction enzymes of the pG1 plasmid, the neor gene, the β-galactosidase gene, the hygromycinr gene, and the SV40 promoter.
The pGene plasmid (FIG. 3) does not exist as an independent molecular entity, but rather may be considered a construction intermediate in the process of cloning genes for subsequent insertion into pG1. The basic backbone is that of pBR322 (Bolivar et al., Gene, 2:95 (1977)). To the 2.1 kb EcoRI/NdeI fragment containing the ampicillin resistance gene and the bacterial origin of replication two linkers were ligated. These linkers, synthesized using an oligo-nucleotide synthesizer, contain a total of 14 unique restriction enzyme recognition sites, as well as sequences felt to enhance mRNA stability and translatability in eukaryotic cells. The restriction sites were chosen based on a screen of non-cutting restriction enzymes of the plasmid backbone, the neor gene, the β-galactosidase gene, the hygromycinr gene, and the SV40 promoter. (The choice of enzymes results in the sites of pG1 being a subset of the sites in pGene). Genes can be ligated into this backbone with NcoI and XhoI ends. The resulting backbone, less the inserted gene, is roughly 2.1 kb in size and contains a 99 base pair multiple cloning site containing, from 5′ to 3′, the following restriction enzyme recognition sites: SphI, NotI, SnaBI, SalI, SacII, AccI, NruI, BgII, NcoI, XhoI, HindIII, ApaI, and SmaI. (FIG. 4). From the BgII to the NcoI sites lies a 27 base pair region containing an mRNA signal based on the work of Hagenbuchle et al. Cell, 13:551-563 (1978). They found that the 3′ terminal sequence of 18S ribosomal RNA is highly conserved among eukaryotes, suggesting that complementary sequences between 18S RNA and mRNA may be involved in positioning the initiating start codon (AUG) on the 30S ribosome. Synthesis of adenovirus 2 late proteins, particularly polypeptide IX, may also follow this rule (Lawrence and Jackson, J. Molec. Biology, 162:317-334) (1982)). Following this ribosomal binding signal, a consensus signal for initiation of translation based on Kozak's rules (Kozak, Nucl. Acids Res., 12:857-872, 1984) was also inserted. We used the wobble at the ATG which permitted use of an NcoI restriction enzyme site.
To make pG1I21SvTk, the plasmid pG1XSvTK was first constructed. First, the Herpes thymidine kinase (TK) gene was subcloned into pBg.
To make pBg, the 3.0 kb BamHI/EcoRI fragment of the lacZ gene encoding β-galactosidase was isolated and two linkers were added. To the 5′ end an NdeI-BamHI linker, containing the 5′ portion of the multiple cloning site up to the NcoI site, as well as the first 21 base pairs of the lacZ gene, was ligated. To the 3′ end, an EcoRI/EcoRI linker completing the 3′ sequence of the lacZ followed by sequence encoding the XhoI, HindIII, ApaI, and SmaI sites was ligated. The sequence of the 5′ EcoRI site was mutated, maintaining amino acid coding fidelity but eliminating the internal EcoRI site to permit directional cloning and screening of the total linkered lacZ fragment into the 2.1 kb NdeI/EcoRI of pBR322. The pBg plasmid was then used to construct an SV40 promoted TK gene. A 1.74 kb Bgl II/PvuII fragment containing the Herpes Virus Type I thymidine kinase gene, was excised from the pX1 plasmid (Huberman et al., Expt1. Cell Res., Vol 153, pgs. 347-362 (1984)), blunted with the large (Klenow) fragment of DNA polymerase I, and inserted into pBg which had first digested with NcoI and XhoI and then also blunted. This resulted in plasmid pTk. The 339 base pair PvuII/HindIII SV40 early promoter fragment was then inserted into pTk which had been digested with SalI and BgIII to generate the plasmid pSvTk. Once pSvTk was obtained, it was a simple matter to obtain the SalI/XhoI fragment containing the SV40-promoted TK gene, blunt it with the large Klenow fragment of DNA polymerase I, and insert it into SalI/HindIII digested and blunted pG1, thereby generating pG1XSvTk.
The plasmid pG1I21SvTk was prepared as follows. The IL-2 gene was derived from the plasmid HT-5.1 (ATCC #59396). The 1.0 kb BamHI fragment was isolated from this plasmid and then truncated down to a 445 base pair HgiAI/DraI fragment. To restore the authentic 5′ coding sequence, a 100 base pair linker was constructed including the entire 20 amino acid coding region of the amino-terminal end of IL-2, and then a 40 base pair stretch identical in sequence to that of pGene between the BglII and NcoI sites is added as a 5′ leader. A SnaBI site was added 5′ to the BglII, permitting direct insertion of this reconstructed IL-2 fragment into pBg which has been digested with SnaBI and HindIII (the HindIII blunted with the Klenow polymerase). From this resulting pI21 plasmid, a 550 base pair BglII/ClaI fragment was isolated blunted, and then inserted into Not I digested and blunted pG1XSvTk.
Generation of Producer Cells
Producer cells for vectors carrying the Herpes simplex virus thymidine kinase gene (TK) as a marker may be generated in two ways. Both methods are developed to generate producer cells containing only a single, provirally integrated vector within the chromosomes of the packaging cell. In the first, the TK gene is co-transfected into PE501 packaging cells (Miller and Rosman, Biotechniques 7:980-990 (1989) with an alternate selectable plasmid, such as pSv2Neo. Once a population of transfected cells is selected based on the neor selectable marker gene of the pSv2Neo plasmid, the PE501 cells are mixed with PA317 amphotropic packaging cells made hygromycin resistant (hygror) with a hygromycin phosphotransferase expressing plasmid, and allowed to culture together for 10-14 days. At the end of this culture period, the PA317 cells, infected with the TK vector packaged in the PE501 cells, are recovered by hygromycin selection. Individual hygror clones are isolated and evaluated for expression of the non-selectable gene (in the above example IL-2), as well as for their ability to transduce target cells. The latter is measured either by the level of non-selectable gene activity that is introduced in target cells, or by direct TK titering using HAT selection (Littlefield, Science 145:709-710 (1964)) of TK- NIH 3T3 fibroblasts in a manner similar to conventional titering with G418 for the neor gene (Eglitis et al, Science 230:1395-1398 (1985)). Briefly, TK- NIH 3T3 cells are plated at a density of 2×104 cells per 35 mm dish and the following day infected for 2-4 hours with various dilutions of virus supernatant containing 8 μg/ml polybrene. The cells are allowed to grow for an additional 24-48 hours following infection and then were grown in selective medium containing HAT for 10-12 days prior to staining with methylene blue and counting individual HAT resistant colonies. Producer clones which generate between 5×104 and 5×105 HAT resistant colony-forming units per ml are then used for further evaluation of efficacy.
A second method utilizes amphotropic packaging cells which are TK-. Having packaging cells which are TK- is important to generate easily trans-infected producer clones of vectors which only contain the TK gene as selectable marker. It is desired to incorporate packaging functions into cells lacking endogenous TK activity so that when TK vector is introduced the resulting vector-containing cells may be directly selected in HAT medium. A means of accomplishing this is to use the pPAM3 packaging system of Miller et al., Mol. Cell. Biol., 5:431-437 (1985). The pPAM3 plasmid, encoding for gag, pol, and env, is transfected into TK- NIH 3T3 cells (Chen and Okayama, Mol. Cell. Biol., 7:2745-2752 (1987) along with a plasmid containing a co-selectable marker such as Sv2Neo (Southern and Berg, J. Molec. Appl. Genet, 1:327-341 (1982)). Cells maximally expressing the env gene are the isolated using antibody specific to env in conjunction with flow cytometry. One can then use this population of high env expressing pPAM3 transfected TK- cells (PAT cells) as the target for trans-infection with vector transiently expressed from an ectotropic packaging cell line 48 hours after high effeciency calcium phosphate transfection. The vector transiently packaged by ectropic packaging cells is used to infect directly the amphotropic PAT cells. Transinfected clones are selected with HAT, and then evaluated as described above.
Example 2
This example describes the construction of pG1T12SvTK, which contains a gene encoding TNF-α. A schematic of the construction of pG1T12SvTK is shown in FIG. 6.
Plasmid pG1XSvTK was constructed as hereinabove described in Example 1. Then, the plasmid pLset5SN was constructed. Plasmid pLset5SN contains a 521 bp NcoI/EcoRI fragment from pTNF-A.BEC (Beckman Catalogue number 267430, and as published in Pennica, et al., Nature, Vol. 312, pg. 724 (1984)) to which had been fused a 51 bp EcoRI/NruI oligomer encoding the natural secretion signal of TNF-α.
To create the full and authentic sequence of TNF-α, a set of 10 oligonucleotides was synthesized and assembled in sets of two or four. The sequence of these oligonucleotides was formulated using the human TNF-α sequence found in Genbank (Accession number X02159). For convenience of cloning, the oligomers were assembled and ligated to the 5′ end of the 521 bp TNF-α fragment from pTNF-A.BEC using the pLXSN plasmid (Miller, et al., Biotechniques, Vol. 7, pgs. 980-991 (1990)). A series of plasmid intermediates, pLSET1SN and pLSET3SN AND LSET4SN, each containing greater assemblages of oligonucleotide fragments, were constructed. To construct pLSET5SN, an 851 bp HindIII fragment from pLSET4SN containing the majority of the TNF-α coding sequence was cloned into the unique HindIII site of pLSET3SN immediately following the assembled sequence for the secretion signal. This ligation resulted in the formation of plasmid pLSET5SN, which contains the entire authentic cDNA sequence of TNF-α, including the sequence of the secretion signal. The entire TNF-α gene containing the natural secretion signal was then removed from pLset5SN as a 730 bp BglII/Bam HI fragment. This fragment was then blunted using the Klenow fragment, and inserted into the unique SnaBI site of pG1XSvTk, to create the final construct pG1T12SvTk.
Example 3
This example describes the construction of pG1T11SvTk, which, like pG1T12SvTk, contains a gene encoding TNF-α. A schematic of the construction of pG1T11SvTk is shown in FIG. 7.
Plasmid pG1XSvTk was constructed as described in Example 1. Then the plasmid pT11 was constructed. The same 521 bp NcoI/EcoRI fragment from pTNF-A.BEC is inserted in place of the lacZ gene in NcoI/EcoRI digested pBG, and an 87 base pair oligomer containing the rat growth hormone secretion signal, as formulated using the rat growth hormone sequence found in Genbank (Accession number J00739; Barta, et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 78, pgs. 4867-4871 (1981)) is then inserted into the NcoI site. The resulting gene is removed as a 608 bp BglII/BamHI fragment, blunted with the Klenow fragment, and inserted into the unique SnaBI site of pG1XSvTk to create pG1T11SvTk.
Example 4
This example describes the construction of pG1TkSvN, a schematic of which is shown in FIG. 8. This vector contains a gene, under the control of an SV40 promoter, which encodes neomycin resistance.
Plasmids pG1 and pBg were constructed as described in Example 1. A 1.74 kb BglII/PvuII fragment was excised from the pX1 plasmid, blunted with the large (Klenow) fragment of DNA polymerase I, and inserted into the unique SnaBI site in the pG1 multiple cloning site, to form plasmid pG1TK.
A 339 bp PvuII/HindIII SV40 early promoter fragment was then inserted into pBg in the unique NruI site to generate the plasmid pSvBg. The pSvBg plasmid was digested with BglII/XhoI to remove the lacZ gene, and the ends were made blunt using the Klenow fragment. An 852 bp EcoRI/AsuII fragment containing the coding sequence of the neomycin resistance gene was removed from pN2 (Armentano, et al., J. Virol., Vol. 61, pgs. 1647-1650 (1987)), blunted with Klenow fragment and ligated into the 2.5 kb blunted BglII/XhoI fragment generated hereinabove, resulting in pSvN. The SV40 promoter/neomycin resistance gene cassette was then removed from pSvN as a 1191 bp SalI/HindIII fragment. The pG1Tk plasmid was then digested with SalI/HindIII and ligated with the SV40/neoR fragment to generate pG1TkSvN. (FIG. 8).
Example 5
This example employs a clone (WP4) (Asher et al., J. Inmunol, Vol. 146, pg. 3227 (1991)) of a murine tumor cell line MCA205 (Wexler et al., J. Natl. Canc. Inst., Vol. 63, pg. 1393 (1979)) for assessing the utility of the HSV-TK gene in a “suicide” system.
WP4 cells are plated in 100 mm tissue culture plates at 1-5×105 cells per plate. After 24 hrs. of culture, growth medium is removed and 10 ml of retroviral supernatant containing pG1TKSvN (104-105 titer) with 8 ug/ml polybrene is added per plate. The cells are incubated from 18 to 24 hrs., and the supernatant is then removed and the cells are placed in growth medium for an additional 48 hours of culture. At the end of this period, the cells are from 80% to 90% confluent. Each 100 mm plate is split into three 100 mm paltes, and the cells are grown as follows:
Plate 1: Expand cells and freeze with no further treatment.
Plate 2: Cells are maintained without treatment as an “unselected” population.
Plate 3: One day after splitting, the cells are placed in 0.4-0.8 mg/ml G418, and the cells are observed daily. When 60% to 70% of the cells have been killed, the selection medium is removed, the culture is washed in PBS, and growth medium is added. The cells are cultured for another 5 to 7 days. Cultures which are 50% to 60% confluent are prepared, and 0.4-0.8 mg/ml of G418 in growth medium is added. The cells are then monitored for growth. After initial cell death, the cells can be maintained in G418 continuously. These cells represent the “selected” population.
Transduced “selected” cells are then plated in 24-well tissue culture plate at 1-2.5×104 cells per well. After 24 hrs. of culture, ganciclovir is added in concentrations ranging from 0.01-100 uM, the cells are monitored for growth and all wells are stained in methylene blue when the control wells (no ganciclovir added) are confluent (at 5 to 7 days). Complete killing of transduced selected cells is observed at concentrations from 0.1 to 1.0 uM of ganciclovir, while untransduced cells are killed at concentrations of 100 uM or greater.
Example 6
WP4 tumor cells form tumors in C57BL/6 or BNX mice when injected subcutaneously. The number of cells required for tumor formation will vary depending of the strain of mouse used. C57BL/6 mice require 5×106-1×107 cells for tumor formation in 10-14 days. BNX mice require 1-5×104 cells for tumor formation in 10-14 days. Tumor cells are suspended in PBS and injected subcutaneously into the hind flank in a 0.5 ml volume. Five to ten days after tumor inoculation, ganciclovir is administered intraperitoneally twice daily for 5 days. A 2-5 mg/ml solution of ganciclovir prepared in serum-free medium (RPMI 1640, DMEM, or other) is used to achieve a dose of 125-150 mg/kg. Animals are monitored and tumor measurements made twice weekly.
At the time of ganciclovir administration, tumors are palpable, but unmeasurable. Tumors arising from untransduced WP4 cells reach a size of 2.0-2.5 cm3 in 40-45 days with or without ganciclovir administration. Tumor cells containing the HSV-TK gene initially form palpable tumors then regress completely and become undetectable within 10-20 days after the start of ganciclovir treatment. Complete, long term tumor ablation is observed in 60-80% of mice having received selected cells plus ganciclovir therapy.
It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.
2 51 bases nucleic acid single linear plasmid DNA 1 AATTCGCGGC CGCTACGTAG TCGACGGATC CCTCCAGAAG CTTGGGCCCA T 51 94 base pairs nucleic acid single linear plasmid DNA Gene may be inserted between base 73 and base 74. 2 CGCATGGCGG CCGCTACGTA GTCGACCCGC GGGTCGACTC GCGAAGATCT TTCCGCAGCA 60 GCCGCCACCA TGGCTCGAGA AGCTTGGGCC CGGG 94

Claims (26)

What is claimed is:
1. A method for terminating expression of a heterologous gene in a mammalian cell of a mammalian host, comprising administering to said mammalian host an agent which kills said mammalian cell by interacting with the gene product of a negative selective marker expressed by said mammalian cell, wherein said mammalian cell comprises a vector sequence comprising said heterologous gene and a gene encoding said negative selective marker.
2. The method according to claim 1, wherein said host is a human.
3. The method according to claim 2, wherein said vector sequence comprises a viral vector sequence.
4. The method according to claim 3, wherein said vector sequence comprises a DNA viral vector sequence.
5. The method according to claim 3, wherein said vector sequence comprises an RNA viral vector sequence.
6. The method according to claim 5, wherein said vector sequence comprises a retroviral vector sequence.
7. The method according to claim 6, wherein said retroviral vector sequence has an incomplete structural gene.
8. The method according to claim 5, wherein said retroviral vector sequence has no intact gag, pol or env gene sequences.
9. The method according to claim 8, wherein said retroviral vector sequence has no gag, pol or env sequences.
10. The method according to claim 2, wherein said heterologous gene encodes a growth factor, a lymphokine, soluble CD4, Factor VIII, Factor IX, ADA, LDL receptor, ApoE, or ApoC.
11. An isolated human cell including an RNA viral vector, said RNA viral vector including a heterologous gene encoding a therapeutic agent and a gene encoding a negative selective marker.
12. The human cell of claim 11 wherein said vector is a retroviral vector.
13. The human cell of claim 12 wherein said retroviral vector has an incomplete structural gene.
14. The human cell of claim 12 wherein said retroviral vector has no intact gag, pol, or env gene sequences.
15. The human cell of claim 14 wherein said retroviral vector has no gag, pol, or env gene sequences.
16. The human cell of claim 11, wherein said therapeutic agent is selected from the group consisting of a growth factor, a lymphokine, soluble CD4, Factor VIII, Factor IX, ADA, LDL receptor, ApoE, and ApoC.
17. The human cell of claim 11 wherein said negative selective market is HSV thymidine kinase.
18. The human cell of claim 16 wherein said therapeutic agent is a growth factor.
19. The human cell of claim 16 wherein said therapeutic agent is a lymphokine.
20. The human cell of claim 16 wherein said therapeutic agent is soluble CD4.
21. The human cell of claim 16 wherein said therapeutic agent is Factor VIII.
22. The human cell of claim 16 wherein said therapeutic agent is Factor IX.
23. The human cell of claim 16 wherein said therapeutic agent is ADA.
24. The human cell of claim 16 wherein said therapeutic agent is the LDL receptor.
25. The human cell of claim 16 wherein said therapeutic agent is ApoE.
26. The human cell of claim 16 wherein said therapeutic agent is ApoC.
US08/289,832 1991-11-14 1994-08-12 Vectors including foreign genes and negative selective markers Expired - Fee Related US6761884B1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US08/289,832 US6761884B1 (en) 1991-11-14 1994-08-12 Vectors including foreign genes and negative selective markers
US08/450,370 US5925345A (en) 1991-11-14 1995-05-25 Vectors including foreign genes and negative selective markers

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US79228191A 1991-11-14 1991-11-14
US08/289,832 US6761884B1 (en) 1991-11-14 1994-08-12 Vectors including foreign genes and negative selective markers

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US79228191A Continuation 1991-11-14 1991-11-14

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US08/450,370 Division US5925345A (en) 1991-11-14 1995-05-25 Vectors including foreign genes and negative selective markers

Publications (1)

Publication Number Publication Date
US6761884B1 true US6761884B1 (en) 2004-07-13

Family

ID=25156356

Family Applications (2)

Application Number Title Priority Date Filing Date
US08/289,832 Expired - Fee Related US6761884B1 (en) 1991-11-14 1994-08-12 Vectors including foreign genes and negative selective markers
US08/450,370 Expired - Fee Related US5925345A (en) 1991-11-14 1995-05-25 Vectors including foreign genes and negative selective markers

Family Applications After (1)

Application Number Title Priority Date Filing Date
US08/450,370 Expired - Fee Related US5925345A (en) 1991-11-14 1995-05-25 Vectors including foreign genes and negative selective markers

Country Status (2)

Country Link
US (2) US6761884B1 (en)
WO (1) WO1993010218A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020127697A1 (en) * 1998-10-01 2002-09-12 University Of Southern California Gene delivery system and methods of use
US20060239990A1 (en) * 2003-04-30 2006-10-26 Nabel Elizabeth G Protein Arginine N-Methyltransferase 2 (PRMT-2)
WO2009032194A1 (en) 2007-08-31 2009-03-12 Whitehead Institute For Biomedical Research Wnt pathway stimulation in reprogramming somatic cells
EP2626416A2 (en) 2007-04-07 2013-08-14 The Whitehead Institute for Biomedical Research Reprogramming of somatic cells
WO2019073055A1 (en) 2017-10-13 2019-04-18 Imba - Institut Für Molekulare Biotechnologie Gmbh Enhanced reprogramming of somatic cells
US11065311B2 (en) 2012-10-25 2021-07-20 Denovo Biopharma Llc Retroviral vector with mini-promoter cassette

Families Citing this family (219)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5716826A (en) * 1988-03-21 1998-02-10 Chiron Viagene, Inc. Recombinant retroviruses
US6241982B1 (en) 1988-03-21 2001-06-05 Chiron Corporation Method for treating brain cancer with a conditionally lethal gene
US6569679B1 (en) 1988-03-21 2003-05-27 Chiron Corporation Producer cell that generates adenoviral vectors encoding a cytokine and a conditionally lethal gene
US6133029A (en) * 1988-03-21 2000-10-17 Chiron Corporation Replication defective viral vectors for infecting human cells
US5662896A (en) 1988-03-21 1997-09-02 Chiron Viagene, Inc. Compositions and methods for cancer immunotherapy
US6337209B1 (en) 1992-02-26 2002-01-08 Glaxo Wellcome Inc. Molecular constructs containing a carcinoembryonic antigen regulatory sequence
WO1993010218A1 (en) * 1991-11-14 1993-05-27 The United States Government As Represented By The Secretary Of The Department Of Health And Human Services Vectors including foreign genes and negative selective markers
US5631236A (en) 1993-08-26 1997-05-20 Baylor College Of Medicine Gene therapy for solid tumors, using a DNA sequence encoding HSV-Tk or VZV-Tk
JPH09504518A (en) * 1993-10-06 1997-05-06 アメリカ合衆国 Treatment of tumors by gene transfer of tumor cells with genes encoding negative selectable markers and cytokines
WO1995014091A2 (en) * 1993-11-18 1995-05-26 Chiron Viagene, Inc. Compositions and methods for utilizing conditionally lethal genes
EP0735887A4 (en) * 1993-12-03 1998-07-15 Genetic Therapy Inc -i(IN UTERO) GENE THERAPY FOR FETUSES
US6451571B1 (en) 1994-05-02 2002-09-17 University Of Washington Thymidine kinase mutants
WO1995030007A1 (en) * 1994-05-02 1995-11-09 University Of Washington Thymidine kinase mutants
ATE381624T1 (en) 1994-05-09 2008-01-15 Oxford Biomedica Ltd RETROVIRAL VECTORS WITH REDUCED RECOMBINATION RATE
US6013517A (en) * 1994-05-09 2000-01-11 Chiron Corporation Crossless retroviral vectors
FR2725213B1 (en) * 1994-10-04 1996-11-08 Rhone Poulenc Rorer Sa VIRAL VECTORS AND USE IN GENE THERAPY
US6638762B1 (en) 1994-11-28 2003-10-28 Genetic Therapy, Inc. Tissue-vectors specific replication and gene expression
US5998205A (en) 1994-11-28 1999-12-07 Genetic Therapy, Inc. Vectors for tissue-specific replication
US5681745A (en) * 1995-05-01 1997-10-28 Trustees Of Boston University Biotin-binding containment systems
FR2751345B1 (en) * 1996-07-16 1998-09-18 Univ Paris Curie HIGHLY PRODUCING PACKAGING LINES
JP2001500738A (en) 1996-09-17 2001-01-23 カイロン コーポレイション Compositions and methods for treating intracellular diseases
US6544523B1 (en) 1996-11-13 2003-04-08 Chiron Corporation Mutant forms of Fas ligand and uses thereof
AU8692598A (en) 1997-08-04 1999-02-22 Calydon, Inc. A human glandular kallikrein enhancer, vectors comprising the enhancer and methods of use thereof
CA2306443A1 (en) 1997-10-14 1999-04-22 Darwin Molecular Corporation Thymidine kinase mutants and fusion proteins having thymidine kinase and guanylate kinase activities
ATE476508T1 (en) 1997-11-06 2010-08-15 Novartis Vaccines & Diagnostic NEISSERIAL ANTIGENS
WO1999036544A2 (en) 1998-01-14 1999-07-22 Chiron S.P.A. Neisseria meningitidis antigens
EP2261349A3 (en) 1998-05-01 2012-01-11 Novartis Vaccines and Diagnostics, Inc. Neisseria meningitidis antigens and compositions
EP1121437B1 (en) 1998-10-15 2008-02-20 Novartis Vaccines and Diagnostics, Inc. Metastatic breast and colon cancer regulated genes
ATE338120T2 (en) 1998-11-27 2006-09-15 Ucb Sa COMPOSITIONS AND METHODS FOR INCREASE BONE MINERALIZATION
US20040009535A1 (en) 1998-11-27 2004-01-15 Celltech R&D, Inc. Compositions and methods for increasing bone mineralization
ES2310055T3 (en) 1998-12-16 2008-12-16 Novartis Vaccines & Diagnostic CYCLIN DEPENDENT HUMAN KINASE (HPNQALRE).
US7063850B1 (en) 1998-12-22 2006-06-20 University Of Tennessee Research Foundation Protective antigen of group A Streptococci
US7935805B1 (en) 1998-12-31 2011-05-03 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV Type C polypeptides, polypeptides and uses thereof
US6602705B1 (en) 1998-12-31 2003-08-05 Chiron Corporation Expression of HIV polypeptides and production of virus-like particles
US6864235B1 (en) 1999-04-01 2005-03-08 Eva A. Turley Compositions and methods for treating cellular response to injury and other proliferating cell disorders regulated by hyaladherin and hyaluronans
US6911429B2 (en) 1999-04-01 2005-06-28 Transition Therapeutics Inc. Compositions and methods for treating cellular response to injury and other proliferating cell disorders regulated by hyaladherin and hyaluronans
WO2000066741A2 (en) 1999-04-30 2000-11-09 Chiron S.P.A. Conserved neisserial antigens
GB9911683D0 (en) 1999-05-19 1999-07-21 Chiron Spa Antigenic peptides
GB9916529D0 (en) 1999-07-14 1999-09-15 Chiron Spa Antigenic peptides
ES2541830T3 (en) 1999-10-29 2015-07-27 Novartis Vaccines And Diagnositics S.R.L. Neisseria antigenic peptides
EP1232264B1 (en) 1999-11-18 2009-10-21 Novartis Vaccines and Diagnostics, Inc. Human fgf-21 gene and gene expression products
EP2275129A3 (en) 2000-01-17 2013-11-06 Novartis Vaccines and Diagnostics S.r.l. Outer membrane vesicle (OMV) vaccine comprising N. meningitidis serogroup B outer membrane proteins
EP1854476A3 (en) 2000-02-09 2008-05-07 Bas Medical, Inc. Use of relaxin to treat diseases related to vasoconstriction
EP2075255A1 (en) 2000-03-08 2009-07-01 Novartis Vaccines and Diagnostics, Inc. Human FGF-23 gene and gene expression products
AU2001263160A1 (en) * 2000-05-19 2001-12-03 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Utilization of non-viral sequences for minus-strand dna transfer and gene reconstitution
EP1950297A2 (en) 2000-05-31 2008-07-30 Novartis Vaccines and Diagnostics, Inc. Compositions and methods for treating neoplastic disease using chemotherapy and radiation sensitizers
US7700359B2 (en) 2000-06-02 2010-04-20 Novartis Vaccines And Diagnostics, Inc. Gene products differentially expressed in cancerous cells
ES2305087T3 (en) 2000-06-15 2008-11-01 Novartis Vaccines And Diagnostics, Inc. POLINUCLEOTIDOS RELATED TO CANCER DE COLON.
CA2425303A1 (en) 2000-10-27 2002-05-02 John Telford Nucleic acids and proteins from streptococcus groups a & b
CA2430379A1 (en) 2000-12-07 2002-06-13 Chiron Corporation Endogenous retroviruses up-regulated in prostate cancer
FR2820146B1 (en) * 2001-01-29 2003-06-27 Centre Nat Rech Scient PROCESS FOR OBTAINING HIGHLY PRODUCING RETROVIRUS ENCAPSIDATION CELLS BY ELIMINATING CELLS PERMISSIVE TO INFECTION BY RETRO VIRUS INFECTION
GB0107661D0 (en) 2001-03-27 2001-05-16 Chiron Spa Staphylococcus aureus
GB0107658D0 (en) 2001-03-27 2001-05-16 Chiron Spa Streptococcus pneumoniae
AU2002303261A1 (en) 2001-04-06 2002-10-21 Georgetown University Gene brcc2 and diagnostic and therapeutic uses thereof
AU2002305151A1 (en) 2001-04-06 2002-10-21 Georgetown University Gene scc-112 and diagnostic and therapeutic uses thereof
WO2002081642A2 (en) 2001-04-06 2002-10-17 Georgetown University Gene brcc-3 and diagnostic and therapeutic uses thereof
EP1967525B1 (en) 2001-05-08 2012-11-14 Darwin Molecular Corporation A method for regulating immune function in primates using the foxp3 protein
US20030198621A1 (en) 2001-07-05 2003-10-23 Megede Jan Zur Polynucleotides encoding antigenic HIV type B and/or type C polypeptides, polypeptides and uses thereof
CA2452015C (en) 2001-07-05 2012-07-03 Chiron Corporation Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof
AU2002365184A1 (en) 2001-10-26 2003-07-30 Id Biomedical Corporation Of Washington Efficient protein expression system
EP1487854B1 (en) 2001-11-09 2010-04-28 Georgetown University Novel isoform of vascular endothelial cell growth inhibitor (vegi)
NZ546711A (en) 2001-12-12 2008-06-30 Chiron Srl Immunisation against chlamydia trachomatis
CA2472282A1 (en) 2002-01-08 2003-07-17 Chiron Corporation Gene products differentially expressed in cancerous breast cells and their methods of use
KR100981471B1 (en) 2002-03-15 2010-09-10 더 큐레이터스 오브 더 유니버시티 오브 미주리 Mutants of the p4 protein of nontypable haemophilus influenzae with reduced enzymatic activity
JP2005520543A (en) 2002-03-21 2005-07-14 サイグレス ディスカバリー， インコーポレイテッド Novel compositions and methods in cancer
US7244565B2 (en) 2002-04-10 2007-07-17 Georgetown University Gene shinc-3 and diagnostic and therapeutic uses thereof
CA2489292A1 (en) 2002-06-13 2003-12-24 Chiron Corporation Vectors for expression of hml-2 polypeptides
UA80447C2 (en) 2002-10-08 2007-09-25 Methods for treating pain by administering nerve growth factor antagonist and opioid analgesic
US7569364B2 (en) 2002-12-24 2009-08-04 Pfizer Inc. Anti-NGF antibodies and methods using same
US9498530B2 (en) 2002-12-24 2016-11-22 Rinat Neuroscience Corp. Methods for treating osteoarthritis pain by administering a nerve growth factor antagonist and compositions containing the same
SI1575517T1 (en) 2002-12-24 2012-06-29 Rinat Neuroscience Corp Anti-ngf antibodies and methods using same
US20070149449A1 (en) 2003-02-14 2007-06-28 Morris David W Therapeutic targets in cancer
US7767387B2 (en) 2003-06-13 2010-08-03 Sagres Discovery, Inc. Therapeutic targets in cancer
US20040170982A1 (en) 2003-02-14 2004-09-02 Morris David W. Novel therapeutic targets in cancer
PL379983A1 (en) 2003-02-19 2006-11-27 Rinat Neuroscience Corp. Methods for treating pain by administering a nerve growth factor antagonist and an nsaid and compositions containing the same
GB0308198D0 (en) 2003-04-09 2003-05-14 Chiron Srl ADP-ribosylating bacterial toxin
US20070178066A1 (en) 2003-04-21 2007-08-02 Hall Frederick L Pathotropic targeted gene delivery system for cancer and other disorders
US8052966B2 (en) 2003-04-21 2011-11-08 University Of Southern California Methods and compositions for treating metastatic cancer
US9532994B2 (en) 2003-08-29 2017-01-03 The Regents Of The University Of California Agents and methods for enhancing bone formation by oxysterols in combination with bone morphogenic proteins
KR20070001932A (en) 2003-12-23 2007-01-04 리나트 뉴로사이언스 코퍼레이션 Agonist anti-trkc antibodies and methods using same
CN104292321A (en) 2004-03-29 2015-01-21 株式会社嘉尔药物 Novel modified galectin 9 protein and use thereof
JP5301152B2 (en) 2004-04-07 2013-09-25 ライナット ニューロサイエンス コーポレイション Method for treating bone cancer pain by administering a nerve growth factor antagonist
ES2673972T3 (en) 2004-07-09 2018-06-26 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Soluble forms of the glycoprotein G of the hendra and nipah viruses
US20060024677A1 (en) 2004-07-20 2006-02-02 Morris David W Novel therapeutic targets in cancer
TWI355389B (en) 2004-07-30 2012-01-01 Rinat Neuroscience Corp Antibodies directed against amyloid-beta peptide a
JP2008530245A (en) 2005-02-18 2008-08-07 ノバルティス ヴァクシンズ アンド ダイアグノスティクス， インコーポレイテッド Antigens from uropathogenic strains
JP2008535494A (en) 2005-04-07 2008-09-04 サグレシュ ディスカバリー， インコーポレイテッド Cancer-related gene (PRLR)
WO2006110599A2 (en) 2005-04-07 2006-10-19 Novartis Vaccines And Diagnostics Inc. Cacna1e in cancer diagnosis, detection and treatment
KR20080039929A (en) 2005-07-22 2008-05-07 와이스 테라퓨틱스 가부시키가이샤 Anti-cd26 antibodies and methods of use thereof
DK3045182T3 (en) 2005-11-14 2018-03-19 Teva Pharmaceuticals Int Gmbh ANTAGONIST ANTIBODIES AGAINST CALCITONIN GEN-RELATED PEPTIDE TO TREAT HEAT
TW200808352A (en) * 2006-02-02 2008-02-16 Rinat Neuroscience Corp Methods for treating unwanted weight loss or eating disorders by administering a TRKB agonist
JP2009525319A (en) * 2006-02-02 2009-07-09 ライナット ニューロサイエンス コーポレイション Method of treating obesity by administering a trkB antagonist
EP1993558B1 (en) 2006-02-27 2014-07-30 The Regents of The University of California Oxysterol compounds and the hedgehog pathway
CN103709252B (en) 2006-06-07 2016-12-07 生物联合公司 Identify antibody and the using method thereof of the sugary epi-position of CD-43 and CEA expressed on cancerous cell
US20100015168A1 (en) 2006-06-09 2010-01-21 Novartis Ag Immunogenic compositions for streptococcus agalactiae
US20100166788A1 (en) 2006-08-16 2010-07-01 Novartis Vaccines And Diagnostics Immunogens from uropathogenic escherichia coli
EP2139447A2 (en) 2007-03-20 2010-01-06 Harold Brem Gm-csf cosmeceutical compositions and methods of use thereof
EP2155777A2 (en) 2007-04-10 2010-02-24 The Administrators of the Tulane Educational Fund Soluble and membrane-anchored forms of lassa virus subunit proteins
GB0714963D0 (en) 2007-08-01 2007-09-12 Novartis Ag Compositions comprising antigens
CA2707663C (en) 2007-12-03 2017-05-30 The Regents Of The University Of California Oxysterols for activation of hedgehog signaling, osteoinduction, antiadipogenesis, and wnt signaling
JP5737944B2 (en) 2007-12-17 2015-06-17 ファイザー・リミテッドＰｆｉｚｅｒ Ｌｉｍｉｔｅｄ Treatment of interstitial cystitis
SG186669A1 (en) 2007-12-18 2013-01-30 Bioalliance Cv Antibodies recognizing a carbohydrate containing epitope on cd-43 and cea expressed on cancer cells and methods using same
EP2274437B1 (en) 2008-04-10 2015-12-23 Cell Signaling Technology, Inc. Compositions and methods for detecting egfr mutations in cancer
WO2009150623A1 (en) 2008-06-13 2009-12-17 Pfizer Inc Treatment of chronic prostatitis
TWI516501B (en) 2008-09-12 2016-01-11 禮納特神經系統科學公司 Pcsk9 antagonists
US20110287026A1 (en) 2008-09-23 2011-11-24 President And Fellows Of Harvard College Sirt4 and uses thereof
US9181315B2 (en) 2009-01-08 2015-11-10 Dana-Farber Cancer Institute, Inc. Compositions and methods for induced brown fat differentiation
WO2010086828A2 (en) 2009-02-02 2010-08-05 Rinat Neuroscience Corporation Agonist anti-trkb monoclonal antibodies
AU2010220103A1 (en) 2009-03-06 2011-09-22 Novartis Ag Chlamydia antigens
WO2010118243A2 (en) 2009-04-08 2010-10-14 Genentech, Inc. Use of il-27 antagonists to treat lupus
SG175092A1 (en) 2009-04-14 2011-11-28 Novartis Ag Compositions for immunising against staphylococcus aerus
WO2010146511A1 (en) 2009-06-17 2010-12-23 Pfizer Limited Treatment of overactive bladder
ES2526996T3 (en) 2009-07-16 2015-01-19 Novartis Ag Detoxified immunogens from Escherichia coli
US8563261B2 (en) 2009-09-28 2013-10-22 The Trustees Of The University Of Pennsylvania Method of diagnosing and treating interstitial cystitis
GB0919690D0 (en) 2009-11-10 2009-12-23 Guy S And St Thomas S Nhs Foun compositions for immunising against staphylococcus aureus
CA2787628A1 (en) 2010-01-21 2011-07-28 Dana-Farber Cancer Institute, Inc. Context specific genetic screen platform to aid in gene discovery and target validation
US8298535B2 (en) 2010-02-24 2012-10-30 Rinat Neuroscience Corp. Anti-IL-7 receptor antibodies
GB201003333D0 (en) 2010-02-26 2010-04-14 Novartis Ag Immunogenic proteins and compositions
PE20130393A1 (en) 2010-03-11 2013-04-07 Rinat Neuroscience Corp ANTIBODIES WITH PH-DEPENDENT ANTIGEN UNION
GB201005625D0 (en) 2010-04-01 2010-05-19 Novartis Ag Immunogenic proteins and compositions
WO2011133931A1 (en) 2010-04-22 2011-10-27 Genentech, Inc. Use of il-27 antagonists for treating inflammatory bowel disease
US8747844B2 (en) 2010-07-30 2014-06-10 Saint Louis University Methods of treating pain
US9539427B2 (en) 2010-11-08 2017-01-10 The Johns Hopkins University Methods for improving heart function
WO2012072769A1 (en) 2010-12-01 2012-06-07 Novartis Ag Pneumococcal rrgb epitopes and clade combinations
EP2663868A2 (en) 2010-12-01 2013-11-20 The University of North Carolina at Chapel Hill Methods and compositions for targeting sites of neovascular growth
US20130071375A1 (en) 2011-08-22 2013-03-21 Saint Louis University Compositions and methods for treating inflammation
WO2013028527A1 (en) 2011-08-23 2013-02-28 Indiana University Research And Technology Corporation Compositions and methods for treating cancer
CA2848368C (en) 2011-09-13 2023-02-14 Dana-Farber Cancer Institute, Inc. Compositions and methods for brown fat induction and activity using fndc5
WO2013055911A1 (en) 2011-10-14 2013-04-18 Dana-Farber Cancer Institute, Inc. Znf365/zfp365 biomarker predictive of anti-cancer response
SG11201401699WA (en) 2011-11-11 2014-09-26 Rinat Neuroscience Corp Antibodies specific for trop-2 and their uses
WO2013093693A1 (en) 2011-12-22 2013-06-27 Rinat Neuroscience Corp. Staphylococcus aureus specific antibodies and uses thereof
EP2794659A1 (en) 2011-12-22 2014-10-29 Rinat Neuroscience Corp. Human growth hormone receptor antagonist antibodies and methods of use thereof
PE20142406A1 (en) 2012-05-04 2015-01-23 Pfizer ANTIGENS ASSOCIATED WITH PROSTATE AND VACCINE-BASED IMMUNOTHERAPY REGIMES
CA2872751A1 (en) 2012-05-07 2013-11-14 The Regents Of The University Of California Oxysterol analogue oxy133 induces osteogenesis and hedgehog signaling and inhibits adipogenesis
US8603470B1 (en) 2012-08-07 2013-12-10 National Cheng Kung University Use of IL-20 antagonists for treating liver diseases
AU2013343099A1 (en) 2012-11-09 2015-05-14 Pfizer Inc. Platelet-derived growth factor B specific antibodies and compositions and uses thereof
CN105246337B (en) 2013-03-14 2019-02-15 吉恩维沃公司 Thymidine kinase diagnostic analysis for gene therapy application
CN105051068A (en) 2013-03-15 2015-11-11 戴埃克斯有限公司 Anti-plasma kallikrein antibodies
JP2016517888A (en) 2013-05-02 2016-06-20 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Oxysterol bone targeting drug for osteoselective bone formation
RU2015147721A (en) 2013-05-07 2017-06-15 Ринат Нейросаенз Корпорэйшн ANTIBODIES AGAINST GLUCAGON RECEPTOR AND METHODS OF USING THEM
EP3044314B1 (en) 2013-07-12 2019-04-10 SeNa Research, Inc. Methods and compositions for interference with dna polymerase and dna synthesis
US10208125B2 (en) 2013-07-15 2019-02-19 University of Pittsburgh—of the Commonwealth System of Higher Education Anti-mucin 1 binding agents and uses thereof
AR097102A1 (en) 2013-08-02 2016-02-17 Pfizer ANTI-CXCR4 ANTIBODY AND ANTIBODY AND DRUG CONJUGATES
WO2015073580A1 (en) 2013-11-13 2015-05-21 Pfizer Inc. Tumor necrosis factor-like ligand 1a specific antibodies and compositions and uses thereof
WO2015087187A1 (en) 2013-12-10 2015-06-18 Rinat Neuroscience Corp. Anti-sclerostin antibodies
WO2015109212A1 (en) 2014-01-17 2015-07-23 Pfizer Inc. Anti-il-2 antibodies and compositions and uses thereof
PE20161439A1 (en) 2014-03-21 2017-01-26 Teva Pharmaceuticals Int Gmbh ANTIBODIES ANTAGONISTS DIRECTED AGAINST THE PEPTIDE RELATED TO THE CALCITONIN GENE AND METHODS USING THE SAME
US11624093B2 (en) 2014-04-24 2023-04-11 Dana-Farber Cancer Institute, Inc. Tumor suppressor and oncogene biomarkers predictive of anti-immune checkpoint inhibitor response
US10308697B2 (en) 2014-04-30 2019-06-04 President And Fellows Of Harvard College Fusion proteins for treating cancer and related methods
ES2913205T3 (en) 2014-05-13 2022-06-01 Bioatla Inc Conditionally active biological proteins
US9840553B2 (en) 2014-06-28 2017-12-12 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
US11111288B2 (en) 2014-08-28 2021-09-07 Bioatla, Inc. Conditionally active chimeric antigen receptors for modified t-cells
AU2015308818B2 (en) 2014-08-28 2021-02-25 Bioatla Llc Conditionally active chimeric antigen receptors for modified T-cells
JP2017532019A (en) 2014-09-03 2017-11-02 バイオアトラ、エルエルシー Discovery and production of conditionally active biological proteins in the same eukaryotic cell production host
SG10201902168PA (en) 2014-09-09 2019-04-29 Unum Therapeutics Chimeric receptors and uses thereof in immune therapy
AU2015328411C1 (en) 2014-10-06 2022-03-03 Dana-Farber Cancer Institute, Inc. Angiopoietin-2 biomarkers predictive of anti-immune checkpoint response
AU2015328163B2 (en) 2014-10-09 2020-10-15 Dana-Farber Cancer Institute, Inc. Multiple-variable IL-2 dose regimen for treating immune disorders
TWI595006B (en) 2014-12-09 2017-08-11 禮納特神經系統科學公司 Anti-pd-1 antibodies and methods of use thereof
CA2977532A1 (en) 2015-03-06 2016-09-15 Dana-Farber Cancer Institute, Inc. Pd-l2 biomarkers predictive of pd-1 pathway inhibitor responses in esophagogastric cancers
US9758575B2 (en) 2015-04-06 2017-09-12 Yung Shin Pharmaceutical Industrial Co. Ltd. Antibodies which specifically bind to canine vascular endothelial growth factor and uses thereof
TWI787645B (en) 2015-04-13 2022-12-21 美商輝瑞股份有限公司 Cd3-specific antibodies, therapeutic bispecific antibodies and their uses
WO2017015334A1 (en) 2015-07-21 2017-01-26 Saint Louis University Compositions and methods for diagnosing and treating endometriosis-related infertility
CN108137705B (en) 2015-07-21 2022-11-08 武田药品工业株式会社 Monoclonal antibody inhibitor of factor XIIA
WO2017015619A1 (en) 2015-07-23 2017-01-26 The Regents Of The University Of California Antibodies to coagulation factor xia and uses thereof
IL302353A (en) 2015-08-19 2023-06-01 Pfizer Tissue factor pathway inhibitor antibodies and uses thereof
EP3350220B1 (en) 2015-09-15 2021-05-19 Scholar Rock, Inc. Anti-pro/latent-myostatin antibodies and uses thereof
EP3362472B1 (en) 2015-10-16 2023-08-30 The Trustees of Columbia University in the City of New York Compositions and methods for inhibition of lineage specific antigens
US11207393B2 (en) 2015-10-16 2021-12-28 President And Fellows Of Harvard College Regulatory T cell PD-1 modulation for regulating T cell effector immune responses
US10138298B2 (en) 2015-10-23 2018-11-27 The Regents Of The University Of California Anti-IL-2 antibodies and compositions and uses thereof
WO2017075037A1 (en) 2015-10-27 2017-05-04 Scholar Rock, Inc. Primed growth factors and uses thereof
US20210106661A1 (en) 2015-10-29 2021-04-15 Dana-Farber Cancer Institute, Inc. Methods for identification, assessment, prevention, and treatment of metabolic disorders using pm20d1 and n-lipidated amino acids
CN106699889A (en) 2015-11-18 2017-05-24 礼进生物医药科技（上海）有限公司 PD-1 resisting antibody and treatment application thereof
RU2744860C2 (en) 2015-12-30 2021-03-16 Кодиак Сайенсиз Инк. Antibodies and their conjugates
ES2904593T3 (en) 2016-01-21 2022-04-05 Pfizer Monospecific and bispecific antibodies to epidermal growth factor receptor variant III and CD3 and their uses
US20210309965A1 (en) 2016-03-21 2021-10-07 Dana-Farber Cancer Institute, Inc. T-cell exhaustion state-specific gene expression regulators and uses thereof
IL308504A (en) 2016-05-13 2024-01-01 Bioatla Llc Anti-ror2 antibodies, antibody fragments, their immunoconjugates and uses thereof
JP7099967B2 (en) 2016-07-01 2022-07-12 リサーチ ディベロップメント ファウンデーション Elimination of Proliferative Cells from Stem Cell-Derived Grafts
US11136409B2 (en) 2016-09-20 2021-10-05 Dana-Farber Cancer Institute, Inc. Compositions and methods for identification, assessment, prevention, and treatment of AML using USP10 biomarkers and modulators
US20180119141A1 (en) 2016-10-28 2018-05-03 Massachusetts Institute Of Technology Crispr/cas global regulator screening platform
WO2018148246A1 (en) 2017-02-07 2018-08-16 Massachusetts Institute Of Technology Methods and compositions for rna-guided genetic circuits
US11198735B2 (en) 2017-03-03 2021-12-14 Rinat Neuroscience Corp. Anti-GITR antibodies and methods of use thereof
US20200056190A1 (en) 2017-03-16 2020-02-20 Pfizer Inc. Tyrosine prototrophy
CA3059938A1 (en) 2017-04-14 2018-10-18 Kodiak Sciences Inc. Complement factor d antagonist antibodies and conjugates thereof
CA3006798A1 (en) 2017-06-02 2018-12-02 Pfizer Inc. Antibodies specific for flt3 and their uses
WO2018226685A2 (en) 2017-06-06 2018-12-13 Dana-Farber Cancer Institute, Inc. Methods for sensitizing cancer cells to t cell-mediated killing by modulating molecular pathways
GB2576680B (en) 2017-06-13 2022-03-23 Bostongene Corp Systems and methods for generating, visualizing and classifying molecular functional profiles
WO2019016784A1 (en) 2017-07-21 2019-01-24 Universidade De Coimbra Anti-nucleolin antibody
EP3692370A2 (en) 2017-10-04 2020-08-12 OPKO Pharmaceuticals, LLC Articles and methods directed to personalized therapy of cancer
AU2019215075A1 (en) 2018-02-01 2020-08-20 Pfizer Inc. Antibodies specific for CD70 and their uses
SG11202006883QA (en) 2018-02-01 2020-08-28 Pfizer Chimeric antigen receptors targeting cd70
KR20200128116A (en) 2018-02-28 2020-11-11 화이자 인코포레이티드 IL-15 variants and uses thereof
EP3797121A1 (en) 2018-05-23 2021-03-31 Pfizer Inc Antibodies specific for cd3 and uses thereof
CA3100829A1 (en) 2018-05-23 2019-11-28 Pfizer Inc. Antibodies specific for gucy2c and uses thereof
BR112021003670A2 (en) 2018-08-28 2021-05-18 Vor Biopharma, Inc. genetically modified hematopoietic stem cells and their uses
US20210005283A1 (en) 2019-07-03 2021-01-07 Bostongene Corporation Techniques for bias correction in sequence data
US20240050529A1 (en) 2019-10-07 2024-02-15 University Of Virginia Patent Foundation Modulating lymphatic vessels in neurological disease
CA3157509A1 (en) 2019-10-10 2021-04-15 Kodiak Sciences Inc. Methods of treating an eye disorder
EP4041773A1 (en) 2019-10-11 2022-08-17 Beth Israel Deaconess Medical Center, Inc. Anti-tn antibodies and uses thereof
US20230067811A1 (en) 2020-01-24 2023-03-02 University Of Virginia Patent Foundation Modulating lymphatic vessels in neurological disease
WO2021205325A1 (en) 2020-04-08 2021-10-14 Pfizer Inc. Anti-gucy2c antibodies and uses thereof
WO2021224850A1 (en) 2020-05-06 2021-11-11 Crispr Therapeutics Ag Mask peptides and masked anti-ptk7 antibodies comprising such
JP2023533793A (en) 2020-07-17 2023-08-04 ファイザー・インク Therapeutic antibodies and their uses
CA3190227A1 (en) 2020-07-30 2022-02-03 Pfizer Inc. Cells having gene duplications and uses thereof
EP4343004A2 (en) 2020-10-19 2024-03-27 Dana-Farber Cancer Institute, Inc. Germline biomarkers of clinical response and benefit to immune checkpoint inhibitor therapy
WO2022104104A2 (en) 2020-11-13 2022-05-19 Dana-Farber Cancer Institute, Inc. Personalized fusion cell vaccines
US20240029829A1 (en) 2020-12-04 2024-01-25 Bostongene Corporation Hierarchical machine learning techniques for identifying molecular categories from expression data
WO2022159793A2 (en) 2021-01-25 2022-07-28 Dana-Farber Cancer Institute, Inc. Methods and compositions for identifying neuroendocrine prostate cancer
US20220372580A1 (en) 2021-04-29 2022-11-24 Bostongene Corporation Machine learning techniques for estimating tumor cell expression in complex tumor tissue
WO2022261183A2 (en) 2021-06-08 2022-12-15 Dana-Farber Cancer Institute, Inc. Compositions and methods for treating and/or identifying an agent for treating intestinal cancers
WO2023012627A1 (en) 2021-08-02 2023-02-09 Pfizer Inc. Improved expression vectors and uses thereof
WO2023049933A1 (en) 2021-09-27 2023-03-30 Sotio Biotech Inc. Chimeric receptor polypeptides in combination with trans metabolism molecules that re-direct glucose metabolites out of the glycolysis pathway and therapeutic uses thereof
WO2023091909A1 (en) 2021-11-16 2023-05-25 Sotio Biotech Inc. Treatment of myxoid/round cell liposarcoma patients
WO2023097119A2 (en) 2021-11-29 2023-06-01 Dana-Farber Cancer Institute, Inc. Methods and compositions to modulate riok2
US20230245479A1 (en) 2022-01-31 2023-08-03 Bostongene Corporation Machine learning techniques for cytometry
WO2023148598A1 (en) 2022-02-02 2023-08-10 Pfizer Inc. Cysteine prototrophy
WO2023158732A1 (en) 2022-02-16 2023-08-24 Dana-Farber Cancer Institute, Inc. Methods for decreasing pathologic alpha-synuclein using agents that modulate fndc5 or biologically active fragments thereof
WO2024015561A1 (en) 2022-07-15 2024-01-18 Bostongene Corporation Techniques for detecting homologous recombination deficiency (hrd)
WO2024040207A1 (en) 2022-08-19 2024-02-22 Sotio Biotech Inc. Genetically engineered natural killer (nk) cells with chimeric receptor polypeptides in combination with trans metabolism molecules and therapeutic uses thereof
WO2024040208A1 (en) 2022-08-19 2024-02-22 Sotio Biotech Inc. Genetically engineered immune cells with chimeric receptor polypeptides in combination with multiple trans metabolism molecules and therapeutic uses thereof

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989009271A1 (en) 1988-03-21 1989-10-05 Viagene, Inc. Recombinant retroviruses
WO1990001547A1 (en) 1988-08-08 1990-02-22 The Upjohn Company Methods for isolating herpes virus thymidine kinase-encoding dna
WO1990007936A1 (en) * 1989-01-23 1990-07-26 Chiron Corporation Recombinant therapies for infection and hyperproliferative disorders
EP0415731A2 (en) * 1989-08-30 1991-03-06 The Wellcome Foundation Limited Novel entities for cancer therapy
WO1991006658A2 (en) * 1989-10-24 1991-05-16 Cetus Corporation Infective protein delivery system
WO1991010728A1 (en) * 1990-01-19 1991-07-25 The United States Government As Represented By The Secretary Of The Department Of Health And Human Services Novel retroviral vectors
WO1991010729A1 (en) * 1990-01-15 1991-07-25 Plant Science Limited Novel peroxidase and its production
WO1992008796A1 (en) 1990-11-13 1992-05-29 Immunex Corporation Bifunctional selectable fusion genes
WO1993002556A1 (en) 1991-07-26 1993-02-18 University Of Rochester Cancer therapy utilizing malignant cells
US5399346A (en) * 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
US5487992A (en) * 1989-08-22 1996-01-30 University Of Utah Research Foundation Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same
US5925345A (en) * 1991-11-14 1999-07-20 Genetic Therapy, Inc. Vectors including foreign genes and negative selective markers

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2074825C (en) * 1990-12-14 2005-04-12 Daniel J. Capon Chimeric chains for receptor-associated signal transduction pathways
US5358866A (en) * 1991-07-03 1994-10-25 The United States Of America As Represented By The Department Of Health And Human Services Cytosine deaminase negative selection system for gene transfer techniques and therapies
GB9115948D0 (en) * 1991-07-24 1991-09-11 Carr Rupert E Marine boom
US5529774A (en) * 1991-08-13 1996-06-25 The Regents Of The University Of California In vivo transfer of the HSV-TK gene implanted retroviral producer cells

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989009271A1 (en) 1988-03-21 1989-10-05 Viagene, Inc. Recombinant retroviruses
WO1990001547A1 (en) 1988-08-08 1990-02-22 The Upjohn Company Methods for isolating herpes virus thymidine kinase-encoding dna
WO1990007936A1 (en) * 1989-01-23 1990-07-26 Chiron Corporation Recombinant therapies for infection and hyperproliferative disorders
US5399346A (en) * 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
US5487992A (en) * 1989-08-22 1996-01-30 University Of Utah Research Foundation Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same
EP0415731A2 (en) * 1989-08-30 1991-03-06 The Wellcome Foundation Limited Novel entities for cancer therapy
WO1991006658A2 (en) * 1989-10-24 1991-05-16 Cetus Corporation Infective protein delivery system
WO1991010729A1 (en) * 1990-01-15 1991-07-25 Plant Science Limited Novel peroxidase and its production
WO1991010728A1 (en) * 1990-01-19 1991-07-25 The United States Government As Represented By The Secretary Of The Department Of Health And Human Services Novel retroviral vectors
WO1992008796A1 (en) 1990-11-13 1992-05-29 Immunex Corporation Bifunctional selectable fusion genes
WO1993002556A1 (en) 1991-07-26 1993-02-18 University Of Rochester Cancer therapy utilizing malignant cells
US5925345A (en) * 1991-11-14 1999-07-20 Genetic Therapy, Inc. Vectors including foreign genes and negative selective markers

Non-Patent Citations (24)

* Cited by examiner, † Cited by third party
Title
B. Gansbacher et al (1991) J. Cellular Biochemistry Suppl 15, Part G, p. 20, Abstract S218.* *
B. Gansbacher et al. (1991). Jour. Cellular Biochemistry Suppl 15, Part G, p. 20, Abstract S218.* *
Borrelli, et al., Nature, vol. 339, pp. 538-541 (Jun. 15, 1989).
Borrelli, et al., PNAS, vol. 85, pp. 7572-7576 (Oct. 1988).
Breakfield, Neurobiology of Aging, vol. 10, pp. 647-648 (1989).
Cox et al. New England Journal of Medicine 299:121-1424, 1978.* *
DW Cox et al. (1978) New England J Medicine 299: 1421-1424.* *
F.L. Moolten et al (1990) Journal of the National Cancer Institute 82 (4): 297-300.* *
F.L. Moolten et al. (1990). Jour. of the National Cancer Institute 82(4): 297-300.* *
H Temin (1990) Human Gene Therapy 1: 111-123.* *
Heyman, et al., PNAS, vol. 86, pp. 2698-2702 (Apr. 1989).
J.A. Zwiebel et al (1990) Ann. N.Y. Acad. Sci. 618: 394-404.* *
J.A. Zwiebel et al. (1990). Ann. N.Y. Acad. Sci. 618: 394-404.* *
J.G. Hellerman et al (1984) Proc. Natl. Acad. Sci. USA 81: 5340-5344.* *
Mahley Science 240:622-630, Apr. 1988.* *
Moolten et al. Journal of the National Cancer Institute 82:297-300, Feb. 1990.* *
Moolten, Cancer Research, vol. 46, pp. 5276-5281 (Oct. 1986).
Moolten, et al., Human Gene Therapy, vol. pp. 125-134 (1990).
Plautz, et al., The New Biologist, vol. 3, No. 7, pp. 709-715 (Jul. 1991).
R.A. Fleischman (1991) Amer. Jour. of Med. Sci. 301 (5): 353-363.* *
R.A. Fleischman (1991) Amer. Jour. of Med. Sci. 301(5): 353-363.* *
RW Mahley (1988) Science 240: 622-630.* *
Selden, et al., Science, vol. 236, pp. 714-718 (May 8, 1991).
Zwiebel et al. Annals of New York Academy of Science 618:394-404, 1990.* *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020127697A1 (en) * 1998-10-01 2002-09-12 University Of Southern California Gene delivery system and methods of use
US8741279B2 (en) 1998-10-01 2014-06-03 University Of Southern California Gene delivery system and methods of use
US20060239990A1 (en) * 2003-04-30 2006-10-26 Nabel Elizabeth G Protein Arginine N-Methyltransferase 2 (PRMT-2)
EP2626416A2 (en) 2007-04-07 2013-08-14 The Whitehead Institute for Biomedical Research Reprogramming of somatic cells
EP3878949A1 (en) 2007-04-07 2021-09-15 Whitehead Institute for Biomedical Research Reprogramming of somatic cells
WO2009032194A1 (en) 2007-08-31 2009-03-12 Whitehead Institute For Biomedical Research Wnt pathway stimulation in reprogramming somatic cells
EP3078738A1 (en) 2007-08-31 2016-10-12 Whitehead Institute for Biomedical Research Wnt pathway stimulation in reprogramming somatic cells
US11065311B2 (en) 2012-10-25 2021-07-20 Denovo Biopharma Llc Retroviral vector with mini-promoter cassette
WO2019073055A1 (en) 2017-10-13 2019-04-18 Imba - Institut Für Molekulare Biotechnologie Gmbh Enhanced reprogramming of somatic cells

Also Published As

Publication number Publication date
WO1993010218A1 (en) 1993-05-27
US5925345A (en) 1999-07-20

Similar Documents

Publication Publication Date Title
US6761884B1 (en) Vectors including foreign genes and negative selective markers
US5672510A (en) Retroviral vectors
EP0953052B1 (en) Crossless retroviral vectors
US6013517A (en) Crossless retroviral vectors
EP0772689B1 (en) Retroviral vectors having a reduced recombination rate
EP0598029B1 (en) Retroviral vectors containing internal ribosome entry sites
AU737801B2 (en) Lentiviral vectors
US6485965B1 (en) Replicating or semi-replicating viral constructs, preparation and uses for gene delivery
Kim et al. Construction of retroviral vectors with improved safety, gene expression, and versatility
WO1995030763A9 (en) Retroviral vectors having a reduced recombination rate
WO1997042338A9 (en) Crossless retroviral vectors
JP3908274B2 (en) Vector having a therapeutic gene encoding an antimicrobial peptide for gene therapy
AU693317B2 (en) Retroviral vectors for expression in embryonic cells
US6190907B1 (en) Retroviral vectors for gene therapy
WO1995009655A1 (en) Treatment of tumors by genetic transformation of tumor cells with genes encoding negative selective markers and cytokines
US5932467A (en) Retroviral vectors pseudotyped with SRV-3 envelope glycoprotein sequences
WO1997012968A9 (en) Retroviral vectors pseudotyped with srv-3 envelope glycoprotein sequences
CA2327444A1 (en) Construction of retroviral producer cells from adenoviral and retroviral vectors
EP0511311A1 (en) Novel retroviral vectors
EP1059356B1 (en) Replicating retroviral constructs, preparation and uses for gene delivery
WO1999036562A1 (en) Nucleotide expression systems with reduced immunogenicity for use in gene therapy
Robbins Retroviral vectors

Legal Events

Date Code Title Description
AS Assignment

Owner name: GENETIC THERAPY, INC., MARYLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MCLACHLIN, JEANNE R.;REEL/FRAME:007352/0797

Effective date: 19950112

AS Assignment

Owner name: UNITED STATES OF AMERICA, THE, AS REPRESENTED BY T

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:FRENCH, ANDERSON W.;REEL/FRAME:007455/0523

Effective date: 19950322

AS Assignment

Owner name: HEALTH AND HUMAN SERVICES, NATIONAL INSTITUTES OF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BLAESE, R. MICHAEL;REEL/FRAME:007554/0381

Effective date: 19950612

AS Assignment

Owner name: GENETIC THERAPY, INC., MARYLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CHIANG, YAWEN L.;REEL/FRAME:007557/0321

Effective date: 19950711

AS Assignment

Owner name: GENETIC THERAPY, INC., MARYLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:EGLITIS, MARTIN;REEL/FRAME:008198/0546

Effective date: 19960925

FPAY Fee payment

Year of fee payment: 4

REMI Maintenance fee reminder mailed
LAPS Lapse for failure to pay maintenance fees
STCH Information on status: patent discontinuation

Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362

FP Lapsed due to failure to pay maintenance fee

Effective date: 20120713